Öljyllä saastuneen maaperän molekulaarinen biomonitorointi ritsoremediaation aikana

Rhizoremediation is the use of microbial populations present in the rhizosphere of plants for environmental cleanup. The idea of this work was that bacteria living in the rhizosphere of a nitrogen-fixing leguminous plant, goat's rue (Galega orientalis), could take part in the degradation of harmful monoaromatic hydrocarbons, such as benzene, toluene and xylene (BTEX), from oil-contaminated soils. In addition to chemical (e.g. pollutant concentration) and physical (e.g. soil structure) information, the knowledge of biological aspects (e.g. bacteria and their catabolic genes) is essential when developing the rhizoremediation into controlled and effective bioremediation practice. Therefore, the need for reliable biomonitoring methods is obvious. The main aims of this thesis were to evaluate the symbiotic G. orientalis - Rhizobium galegae system for rhizoremediation of oil-contaminated soils, to develop molecular methods for biomonitoring, and to apply these methods for studying the microbiology of rhizoremediation. In vitro, Galega plants and rhizobia remained viable in m-toluate concentrations up to 3000 mg/l. Plant growth and nodulation were inhibited in 500 mg/l m-toluate, but were restored when plants were transferred to clean medium. In the greenhouse, Galega showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 mg/l m-toluate. The high aromatic tolerance of R. galegae and the viability of Galega plants in oil-polluted soils proved this legume system to be a promising method for the rhizoremediation of oil-contaminated soils. Molecular biomonitoring methods were designed and/or developed further for bacteria and their degradation genes. A combination of genomic fingerprinting ((GTG)5-PCR), taxonomic ribotyping of 16S rRNA genes and partial 16S rRNA gene sequencing were chosen for molecular grouping of culturable, heterogeneous rhizosphere bacteria. PCR primers specific for the xylE gene were designed for TOL plasmid detection. Amplified enzyme-coding DNA restriction analysis (AEDRA) with AluI was used to profile both TOL plasmids (xylE primers) and, in general, aromatics-degrading plasmids (C230 primers). The sensitivity of the direct monitoring of TOL plasmids in soil was enhanced by nested C23O-xylE-PCR. Rhizosphere bacteria were isolated from the greenhouse and field lysimeter experiments. High genetic diversity was observed among the 50 isolated, m-toluate tolerating rhizosphere bacteria in the form of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. The inoculum Pseudomonas putida PaW85/pWW0 was not found in the rhizosphere samples. Even if there were no ecological niches available for the bioaugmentation bacterium itself, its conjugative catabolic plasmid might have had some additional value for other bacterial species and thus, for rhizoremediation. Only 10 to 20% of the isolated, m-toluate tolerating bacterial strains were also able to degrade m-toluate. TOL plasmids were a major group of catabolic plasmids among these bacteria. The ability to degrade m-toluate by using enzymes encoded by a TOL plasmid was detected only in species of the genus Pseudomonas, and the best m-toluate degraders were these Pseudomonas species. Strain-specific differences in degradation abilities were found for P.oryzihabitans and P. migulae: some of these strains harbored a TOL plasmid - a new finding observed in this work, indicating putative horizontal plasmid transfer in the rhizosphere. One P. oryzihabitans strain harbored the pWW0 plasmid that had probably conjugated from the bioaugmentation Pseudomonas. Some P. migulae and P. oryzihabitans strains seemed to harbor both the pWW0- and the pDK1-type TOL plasmid. Alternatively, they might have harbored a TOL plasmid with both the pWW0- and the pDK1-type xylE gene. The breakdown of m-toluate by gram-negative bacteria was not restricted to the TOL pathway. Also some gram-positive Rhodococcus erythropolis and Arthrobacter aurescens strains were able to degrade m-toluate in the absence of a TOL plasmid. Three aspects of the rhizosphere effect of G. orientalis were manifested in oil-contaminated soil in the field: 1) G. orientalis and Pseudomonas bioaugmentation increased the amount of rhizosphere bacteria. G. orientalis especially together with Pseudomonas bioaugmentation increased the numbers of m-toluate utilizing and catechol positive bacteria indicating an increase in degradation potential. 2) Also the bacterial diversity, when measured as the amount of ribotypes, was increased in the Galega rhizosphere with or without Pseudomonas bioaugmentation. However, the diversity of m-toluate utilizing bacteria did not significantly increase. At the community level, by using the 16S rRNA gene PCR-DGGE method, the highest diversity of species was also observed in vegetated soils compared with non-vegetated soils. Diversified communities may best guarantee the overall success in rhizoremediation by offering various genetic machineries for catabolic processes. 3) At the end of the experiment, no TOL plasmid could be detected by direct DNA analysis in soil treated with both G. orientalis and Pseudomonas. The detection limit for TOL plasmids was encountered indicating decreased amount of degradation plasmids and thus, the success of rhizoremediation. The use of G. orientalis for rhizoremediation is unique. In this thesis new information was obtained about the rhizosphere effect of Galega orientalis in BTEX contaminated soils. The molecular biomonitoring methods can be applied for several purposes within environmental biotechnology, such as for evaluating the intrinsic biodegradation potential, monitoring the enhanced bioremediation, and estimating the success of bioremediation. Environmental protection by using nature's own resources and thus, acting according to the principle of sustainable development, would be both economically and environmentally beneficial for society. Keywords: molecular biomonitoring, genetic fingerprinting, soil bacteria, bacterial diversity, TOL plasmid, catabolic genes, horizontal gene transfer, rhizoremediation, rhizosphere effect, Galega orientalis, aerobic biodegradation, petroleum hydrocarbons, BTEXSuomessa on tilastoitu tuhansia öljyllä saastuneita maa-alueita, kuten hylättyjä huoltoasemia. Näitä voitaisiin puhdistaa kasvien juuristossa elävien mikrobien avulla eli ritsoremediaatiolla. Tässä työssä tutkittiin typpeä sitovan palkokasvin, vuohenherneen juuriston bakteerien osallistumista monoaromaattisten hiilivetyjen, kuten bentseenin, tolueenin ja ksyleenin, hajottamiseen öljyllä saastuneessa maaperässä. Biologisten tekijöiden tuntemus (mm. bakteerit ja niiden hajotusgeenit) on oleellista kemiallisen (esim. myrkkypitoisuus) ja fysikaalisen (esim. maan rakenne) tiedon lisäksi, jotta ritsoremediaatiosta voidaan kehittää hallittu ja tehokas biopuhdistusmenetelmä. Siksi myös luotettavien biologisten seurantamenetelmien kehittämisen tarve on ilmeinen. Tämän väitöskirjatyön tarkoituksena oli 1) arvioida vuohenherneen ja sen ritsobin eli symbioottisen typensitojabakteerin soveltuvuus öljyllä saastuneen maaperän puhdistamiseen, 2) kehittää molekyylitason menetelmiä biomonitorointia varten ja 3) soveltaa näitä menetelmiä tutkittaessa mikrobiologisia ilmiöitä ritsoremediaation aikana. Ritsobin korkea aromaattisten aineiden sietokyky ja vuohenherneen elinkykyisyys öljymaissa osoittivat tämän palkokasvi-bakteeri -yhdistelmän soveltuvan hyvin öljyllä saastuneen maaperän ritsoremediaatioon. Molekyylibiologisia menetelmiä suunniteltiin ja/tai kehitettiin bakteerien ja niiden hajotusgeenien seuraamiseksi maassa. Viljeltävissä olevia, heterogeenisia juuristobakteereita ryhmiteltiin erään vähemmän käytetyn, mutta tässä työssä tehokkaaksi osoitetun, genomisen sormenjälkimenetelmän, taksonomisen ribotyyppauksen ja sekvenoinnin avulla. Yhteisötasolla, ja siten myös ei-viljeltävissä olevien bakteerien havainnoimiseksi, käytettiin denaturoivaa gradienttigeelielektroforeesia. Geenispesifinen PCR-menetelmä suunniteltiin TOL-plasmidien tunnistamiseksi. Tehoste-PCR kehitettiin TOL-plasmidien havaitsemiseksi herkästi suoraan maasta. Sekä TOL-plasmideja että laajemminkin erilaisia aromaatteja hajottavia entsyymejä koodaavia plasmideja profiloitiin DNA:n pilkonta-analyysillä. Vuohenherneen juuristovaikutukset öljyllä saastuneessa maapeässä olivat seuraavat: 1) Vuohenherne lisäsi juuristobakteerien määrää, mutta erityisesti yhdessä TOL-plasmidin sisältävän Pseudomonas-bakteerin kanssa m-toluaattia hajottavien bakteerien lukumäärää. Tämä osoitti hajotuspotentiaalin olevan suurempaa kasvillisissa maissa kuin kasvittomissa maissa. 2) Vuohenherneen juuristossa oli kasvitonta maata enemmän eri bakteerilajeja, mutta ei kuitenkaan merkittävästi enemmän m-toluaattia hajottavia bakteerilajeja. Myös yhteisötasolla suurin bakteerilajien monimuotoisuus havaittiin kasvillisissa maissa. Monipuoliset yhteisöt saattavatkin parhaiten taata tehokkaan ritsoremediaation tarjoamalla lukuisia geneettisiä koneistoja hajotusprosessien käyttöön. 3) Kokeen lopussa TOL-plasmideja ei kyetty enää havaitsemaan suoralla DNA-analyysillä maasta, jossa oli sekä vuohenherne että Pseudomonas. TOL-plasmidien havaintoraja oli siis ylittynyt osoittaen hajotusplasmidien määrän ja siten katabolisen aktiivisuuden vähentyneen. Tämä viittasi ritosremediaation onnistumiseen. 4) Eräältä P. oryzihabitans -kannalta löytyi kokeissa käyttämämme P. putidan TOL-plasmidi. Lisäksi eräiltä P. migulae - ja P. oryzihabitans -kannoilta löydettiin TOL-plasmidi, jota niillä ei tiedetty olevan. Nämä tapahtumat osoittivat mahdollista hajotusplasmidin siirtymistä bakteerikannasta toiseen ja siten puhdistumisen tehostumista vuohenherneen juuristossa. Vuohenherneen käyttö ritsoremediaatiossa on maailmanlaajuisestikin ainutlaatuista. Ympäristönsuojelu luonnon omia voimavaroja käyttäen, ja siten kestävän kehityksen periaatteen mukaisesti toimien, olisi sekä taloudellisesti että viihtyisän ja puhtaan ympäristön säilymisen kannalta hyödyllistä yhteiskunnalle. Tässä väitöskirjatyössä uutta tietoa saatiin vuohenherneen juuristovaikutuksista monoaromaattisilla hiilivedyillä saastuneissa maissa. Hajotusplasmidien liikkuminen bakteerikannasta tai -lajista toiseen osoittautui ilmiöksi, jota kannattaa seurata jatkossa tarkemmin, jotta puhdistumisen aikaisista dynaamisista muutoksista ja geeniekologisista lainalaisuuksista saataisiin enemmän tietoa. Kehitettyjä molekulaarisia biomonitorointimenetelmiä voidaan soveltaa moneen tarkoitukseen ympäristöbioteknologian alalla, kuten maan luontaisen biohajotuspotentiaalin kartoittamiseen, bioremediaation tehostamisen seurantaan ja puhdistusprosessin onnistumisen arviointiin

Potential of the Galega – Rhizobium galegae System for Bioremediation of Oil-Contaminated Soil

Bioremediation potential of the nitrogen-fixing leguminous plant Galega orientalis Lam. and its microsymbiont Rhizobium galegae was evaluated in microcosm and mesocosm scale in oil and BTEX (benzene, toluene, ethylbenzene, xylene) contaminated soils, with m-toluate serving as a model for the latter group. G. orientalis and Rhizobium galegae remained viable in m-toluate fractions up to 3000 ppm. Plant growth and nodulation were inhibited in 500 ppm m-toluate, but were restored when plants were transferred to clean medium. In soil, G. orientalis nodulated and showed good growth in 2000 ppm m-toluate as well as in diesel-contaminated soil in the field, where the plant was stimulating bacterial growth in the rhizosphere. A collection of 52 indigenous m-toluate-tolerating bacteria isolated from oil-contaminated rhizosphere of G. orientalis was characterised and identified by classical and molecular biological methods. 16SrDNA PCR-RFLP and (GTG)5-PCR genomic fingerprinting combined with partial sequencing indicated the presence of five major lineages of the Bacteria domain. A TOL plasmid-specific xylE-PCR was developed in order to detect both active and potential degraders of m-toluate. The ability to degrade m-toluate in the presence of the gene xylE was detected only within the genus Pseudomonas. The isolates were tested for capacity to grow on m-toluate as their sole carbon and energy source. In laboratory experiments, the best rhizosphere isolates performed equally well to the positive control strain and are good candidates for inoculant production in the future. They have been tagged with marker genes for further studies on colonisation and persistence

Preoperative measurements on MRI in Chiari 1 patients fail to predict outcome after decompressive surgery

Abstract Background: The purpose of our study was to research the parameters of magnetic resonance imaging (MRI) that would predict the outcome of surgery in patients with Chiari 1 malformation (CM1) and to evaluate changes in MRI parameters after surgery. Methods: Fifty-one patients (19 children, 13 adolescents, and 19 adults) operated on due to CM1 in Oulu University Hospital between 2004 and 2018 were evaluated. Seventeen parameters were measured from the preoperative MRI and 11 from the postoperative MRI. The correlations between the MRI parameters and the clinical variables before and after surgery were analyzed. Results: The majority (88.2%) of the patients had favorable surgical outcomes. Postoperatively, subjective symptoms improved in 88.6% of the patients and syringomyelia in 81.8%. The location of the cerebellar tonsils, when measured in relation to the C2 synchondrosis or the end plate, postoperatively moved cranially in 51.0% (n = 26), did not change in 27.4% (n = 14), and moved caudally in 21.6% (n = 11) of the patients. However, neither the location of the tonsils nor any other parameters measured from pre- or postoperative MRI correlated with the patients’ symptoms or surgical outcomes. Conclusions: No specific parameters on preoperative MRI evaluation were predictive of the outcome of surgery, emphasizing clinical examination in surgical decision-making. Furthermore, the postoperative MRI parameters did not correlate with the surgical outcomes. Thus, routine postoperative imaging is suggested only for patients with preoperatively diagnosed syringomyelia or worsening of symptoms

Genetic architecture of human plasma lipidome and its link to cardiovascular disease

Abstract Understanding genetic architecture of plasma lipidome could provide better insights into lipid metabolism and its link to cardiovascular diseases (CVDs). Here, we perform genome-wide association analyses of 141 lipid species (n = 2,181 individuals), followed by phenome-wide scans with 25 CVD related phenotypes (n = 511,700 individuals). We identify 35 lipid-species-associated loci (P <5 ×10−8), 10 of which associate with CVD risk including five new loci-COL5A1, GLTPD2, SPTLC3, MBOAT7 and GALNT16 (false discovery rate<0.05). We identify loci for lipid species that are shown to predict CVD e.g., SPTLC3 for CER(d18:1/24:1). We show that lipoprotein lipase (LPL) may more efficiently hydrolyze medium length triacylglycerides (TAGs) than others. Polyunsaturated lipids have highest heritability and genetic correlations, suggesting considerable genetic regulation at fatty acids levels. We find low genetic correlations between traditional lipids and lipid species. Our results show that lipidomic profiles capture information beyond traditional lipids and identify genetic variants modifying lipid levels and risk of CVD

New insights into the genetic etiology of Alzheimer’s disease and related dementias

Characterization of the genetic landscape of Alzheimer’s disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/‘proxy’ AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele

Chromosome Xq23 is associated with lower atherogenic lipid concentrations and favorable cardiometabolic indices

Abstract Autosomal genetic analyses of blood lipids have yielded key insights for coronary heart disease (CHD). However, X chromosome genetic variation is understudied for blood lipids in large sample sizes. We now analyze genetic and blood lipid data in a high-coverage whole X chromosome sequencing study of 65,322 multi-ancestry participants and perform replication among 456,893 European participants. Common alleles on chromosome Xq23 are strongly associated with reduced total cholesterol, LDL cholesterol, and triglycerides (min P = 8.5 × 10−72), with similar effects for males and females. Chromosome Xq23 lipid-lowering alleles are associated with reduced odds for CHD among 42,545 cases and 591,247 controls (P = 1.7 × 10−4), and reduced odds for diabetes mellitus type 2 among 54,095 cases and 573,885 controls (P = 1.4 × 10−5). Although we observe an association with increased BMI, waist-to-hip ratio adjusted for BMI is reduced, bioimpedance analyses indicate increased gluteofemoral fat, and abdominal MRI analyses indicate reduced visceral adiposity. Co-localization analyses strongly correlate increased CHRDL1 gene expression, particularly in adipose tissue, with reduced concentrations of blood lipids

Sleep apnoea is a risk factor for severe COVID-19

Background Obstructive sleep apnoea (OSA) is associated with higher body mass index (BMI), diabetes, older age and male gender, which are all risk factors for severe COVID-19.We aimed to study if OSA is an independent risk factor for COVID-19 infection or for severe COVID-19.Methods OSA diagnosis and COVID-19 infection were extracted from the hospital discharge, causes of death and infectious diseases registries in individuals who participated in the FinnGen study (n=260 405). Severe COVID-19 was defined as COVID-19 requiring hospitalisation. Multivariate logistic regression model was used to examine association. Comorbidities for either COVID-19 or OSA were selected as covariates. We performed a meta-analysis with previous studies.Results We identified 445 individuals with COVID-19, and 38 (8.5%) of them with OSA of whom 19 out of 91 (20.9%) were hospitalised. OSA associated with COVID-19 hospitalisation independent from age, sex, BMI and comorbidities (p-unadjusted=5.13×10−5, OR-adjusted=2.93 (95% CI 1.02 to 8.39), p-adjusted=0.045). OSA was not associated with the risk of contracting COVID-19 (p=0.25). A meta-analysis of OSA and severe COVID-19 showed association across 15 835 COVID-19 positive controls, and n=1294 patients with OSA with severe COVID-19 (OR=2.37 (95% 1.14 to 4.95), p=0.021).Conclusion Risk for contracting COVID-19 was the same for patients with OSA and those without OSA. In contrast, among COVID-19 positive patients, OSA was associated with higher risk for hospitalisation. Our findings are in line with earlier works and suggest OSA as an independent risk factor for severe COVID-19

Evidence of a causal effect of genetic tendency to gain muscle mass on uterine leiomyomata

Uterine leiomyomata (UL) are the most common tumours of the female genital tract and the primary cause of surgical removal of the uterus. Genetic factors contribute to UL susceptibility. To add understanding to the heritable genetic risk factors, we conduct a genome-wide association study (GWAS) of UL in up to 426,558 European women from FinnGen and a previous UL meta-GWAS. In addition to the 50 known UL loci, we identify 22 loci that have not been associated with UL in prior studies. UL-associated loci harbour genes enriched for development, growth, and cellular senescence. Of particular interest are the smooth muscle cell differentiation and proliferation-regulating genes functioning on the myocardin-cyclin dependent kinase inhibitor 1A pathway. Our results further suggest that genetic predisposition to increased fat-free mass may be causally related to higher UL risk, underscoring the involvement of altered muscle tissue biology in UL pathophysiology. Overall, our findings add to the understanding of the genetic pathways underlying UL, which may aid in developing novel therapeutics.Peer reviewe

New insights into the genetic etiology of Alzheimer’s disease and related dementias

Characterization of the genetic landscape of Alzheimer’s disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/‘proxy’ AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele